PROJECT SUMMARY Cell-cell communication is an important component of most functions of the cornea, including fibrosis, regeneration, and homeostasis. To a great extent, cell-cell communication has been thought to consist of the release of numerous soluble growth factors and cytokines to direct corneal wound repair; however an underlying question remains as to how these factors perfuse with any specificity from one cell to an adjacent cell. Recently, interest in another method of cell-communication has boomed. This method involves the secretion and uptake of small vesicles, termed Extracellular Vesicles (EVs). EVs can migrate long and short distances to be taken up by other cells. This crosstalk between cells is notable in that EVs carry ?cargo? consisting of proteins, mRNA, miRNA, and DNA that can influence many of the functions in the recipient cell. Indeed, recent findings indicate that development of cancer involves crosstalk with EVs, where cancer cells produce EVs that act on the host's cells to produce a microenvironment that promotes growth of cancer cells. We propose to examine if corneal wound healing involves a similar mechanism, where epithelial cells release EVs that alter the properties of keratocytes. We have found that epithelial EVs can stimulate keratocytes to become myofibroblasts and that epithelial EVs from wounded cells have an even greater effect. This has lead us to the hypothesis that the epithelial EV cargo is altered as cells become migratory to heal a wound, subsequently causing the keratocyte to become myofibroblasts, which produce a microenvironment that promotes their persistence. Because of its clarity and accessibility, the cornea provides an outstanding model to examine movement of EVs through matrix in in vivo or ex vivo models. Techniques to be used include EV isolation, proteomics. miRNA isolation and characterization, transmission electron microscopy, confocal microscopy, and 3D cultures. Relevance to Public Health?EVs are involved in cell-cell communication in virtually all physiological and pathological processes. Because of their ability to travel long distances and interact with specific cells, EVs are widely being examined for usefulness to deliver therapeutics. We will determine if topical application of EVs to the cornea is useful as a therapeutic approach. Potentially, any corneal injury or disease could be treated with EVs.